1. Field of the Invention
The present invention relates to a method for mass production of taxol from Taxus genus plant, more specifically, to a rapid and simple method for mass production of taxol with a high purity and recovery, which comprises the steps of solvent extraction of biomass from Taxus genus plant employing methanol, dichloromethane and hexane, adsorbent treatment, precipitation in hexane, fractional precipitation and high performance liquid chromatography.
2. Description of the Prior Art
Taxanes are diterpene compounds containing the taxane skeleton. For example, taxol is famous as the first identified compound with a taxane ring which is effective for the treatment of leukemia and cancer. Recently, it has been reported that taxol Xs capable of curing approximately 30%, 50% and 20% of ovarian, breast and lung cancer patients, respectively. Also, taxane compounds include baccatin III, 10-deacetylbaccatin III, 10-deacetyltaxol, cephalomannine and deacetylcephalomannine, which are employed in the semi-synthesis of taxol.
Taxane compounds are represented as general formula (I) and (II) as followings: ##STR1## wherein, if R.sub.1 =AC, the compound is baccatin III; and, if R.sub.1 =OH, the compound is 10-deacetylbaccatin III. ##STR2## wherein, if R.sub.2 =AC, R.sub.3 =C.sub.6 H.sub.5, the compound is taxol; if R.sub.2 =OH, R.sub.3 =C.sub.6 H.sub.5, the compound is 10-deacetyltaxol;
if R.sub.2 =AC, R.sub.3 =CH.sub.3 CH.dbd.CH(CH.sub.3), the compound is cephalomannine; and, PA2 if R.sub.2 =OH, R.sub.3 =CH.sub.3 CH.dbd.CH(CH.sub.3), the compound is 10-deacetylcephalomannine.
On the other hand, total synthesis, semi-synthesis and extraction methods have been employed to prepare taxol.
The total synthesis method, however, has not been practically applied in the art, since it requires very expensive chemical reagents and the yield is not so high, which can be expected from the complicated chemical structure of taxol.
The semi-synthesis method employing precursors such as 10-deacetylbaccatin III, has revealed some drawbacks since it essentially entails complicated and multiple steps of isolating and purifying taxol precursors from Taxus genus plant and transforming the taxol precursors to taxol, which, in turn, has been an obstacle to the universal use of the method.
Accordingly, extraction methods by which taxol can be isolated from Taxus genus plants in a direct manner, have prevailed in the art, since they have the advantage of economy, and a variety of approaches have been described in the art:
WO 94/12268 discloses a method of isolating taxol by employing a semi-permeable membrane and reverse osmosis apparatus. However, said method has revealed a serious problem that it essentially requires the expensive semi-permeable membrane and reverse osmosis apparatus accompanied by complicated techniques for operating them.
EP 553,780 A teaches a method of isolating taxol and precursor thereofs which comprises the steps of vacuum drying a methanol extract of Taxus genus plant, solvent extraction employing cyclohexane and methylenechloride to give crude taxol followed by silica gel column HPLC.
WO 92/18492 describes a method of purifying taxol which comprises the steps of methanol extraction of Taxus genus plant, partitional fractionation employing methylenechloride or ethylacetate to obtain crude taxol and normal-phase liquid chromatography.
WO 92/07842 illustrates a method isolating taxol which comprises the steps of a series of solvent extractions employing ethanol, chloroform and methanol to give crude taxol followed by reverse-phase HPLC.
WO 94/13827 suggests a method of purifying taxol which comprises the steps of organic solvent extraction using ethanol, methanol and acetone, adsorbent treatment of activated carbon or charcoal to give crude taxol followed by normal-phase liquid chromatography.
JP 6-157329 A offers a method of obtaining crude taxol of low-purity which comprises a series of solvent extractions employing ethylacetate, ether, acetonitrile and acetone.
However, all of the prior art purification methods, which primarily aim to obtain crude taxol of low-purity by employing solvent extraction and chromatography works, essentially provide taxol-related compounds such as terpenoids, lipids, chlorophyll and phenols, accompanying the taxol of interest. Accordingly, high-purity of taxol has not been obtained even in the case of employing so many chromatographic columns, leading to a heavy load of impurities on the columns used in the purification steps.
Moreover, since the purity of taxol thus obtained is not high, solubility in organic solvent is naturally so low that recovery and yield in the course of chromatography can not be controlled and extra steps for crystallization are essentially required to obtain high-purity crystallized taxol. Accordingly, the prior art purification methods have not been practically employed in the art and there has been a continuous need in the art to develop a method for isolating high-purity taxol in a more simple and economical manner.